We believe this site might serve you best:

United States

Select a Different Country and Language

Promega's Cookie Policy

Our website uses functional cookies that do not collect any personal information or track your browsing activity. When you select your country, you agree that we can place these functional cookies on your device.

What can I do to prevent star activity in my restriction enzyme digestions?

SEARCH ARTICLES

Related Products

Related Resources

LabFact #11

New restriction enzyme isoschizomers often are less expensive or offer performance advantages over the classic enzymes. For more information on the use of isoschizomers and neoschizomers, see the Promega Notes article, "Making the Cut...".

Related Articles

Any restriction endonuclease can be subject to star activity. Generally, star activity (i.e., the enzyme cuts a sequence that is an imperfect match to its known target) is caused by nonoptimal reaction conditions.(1)
,(2)
These include:

High concentration of glycerol (>5% v/v).

High ratio of enzyme units to micrograms of DNA. This ratio depends on each enzyme but generally means >10 units per microgram DNA.

Substitution of other divalent cations [e.g., Mn2+, Cu2+, Co2+ or Zn2+] for Mg2+.

To prevent star activity, we recommend the following guidelines:

Use as few units of restriction enzyme as possible for a complete digestion, which avoids overdigestion of the DNA and reduces the final glycerol concentration in the reaction. We recommend using one unit of restriction enzyme for every microgram of DNA and incubating the reaction for 1 hour. Note: Supercoiled DNA requires three- to fivefold more enzyme than linear DNA.

Make sure the reaction is free of any organic solvents such as alcohols, which may have carried over during DNA purification.

Increase the ionic strength of the reaction buffer to 50–150mM as long as the enzyme is not inhibited by high salt.

Keep the pH in the range of 7.2–8.5, depending on the restriction endonuclease.

Scientists at Your Service

We offer a range of services to help you succeed using Promega technologies. From product training to set up of automated systems and development of custom applications—our scientific support goes beyond the basics.